What is the maximum binding capacity for proteins on
the MaxiSorp surface and the PolySorp surface?
Molecules bind to the PolySorp and MaxiSorp surfaces through passive adsorption.
Using IgG as a reference molecule and knowing that it is a globular molecule,
theoretical calculations indicate that the maximum binding for the MaxiSorp
surface, in monolayer, is 650 ng/cm2. For the PolySorp
surface, the binding capacity is 220 ng/cm2. A
detailed discussion of the principles and calculations is presented in Nunc
Bulletin No. 6. See Nunc Bulletin No. 6.
Which 96-well Nunc-Immuno Plates or Modules are
appropriate for which application?
The following list offers a brief description of the features of Nunc products
and their specific applications.
What is the method for binding streptavidin followed
by an addition of a biotinylated antibody using Nunc-Immuno Plates with MaxiSorp
surface?
Streptavidin has a very low affinity to polystyrene and is most commonly
immobilized using the protein-avian-biotin-capture (PABC) system. In this
system, streptavidin acts as a bridge between the capture antibody and an
irrelevant, biotinylated protein which is adsorbed to a solid phase.
Streptavidin bound directly to plastic surfaces is impaired in both its ability
to bind to biotinylated monoclonal antibody and in its functional affinity when
compared to streptavidin used in the PABC system. Therefore, it is not
recommended to use streptavidin hydrophobically adsorbed to solid phase to
immobilize biotinylated molecules. Passive adsorption of streptavidin to Nunc
MaxiSorp plates can be achieved by using conventional methods: 0.1 mg/ml,
carbonate buffer (pH 9.6) with overnight incubation at room temperature. To
optimize some use a buffer with a pH of 5 or 6.
When performing ELISA using Nunc-Immuno MaxiSorp
Plates, what are some recommendations for reducing high background readings and
non-specific binding?
Assay sensitivity depends strongly on an efficient removal of non-specific
reacting molecules. High background readings and coating instability can be
eliminated by addition of a blocking step after the first coating. The excess
surface is then occupied by indifferent molecules. We recommend washing three
times after each coating step by using a solution of 0.15 M phosphate buffer (pH
7.2) with 0.2 M NaCl and 0.05% Tween 20. For blocking, we recommend using 0.5%
BSA, 1% casein or 1% gelatin in 0.15 M phosphate buffer (pH 8.2) or carbonate
buffer (pH 9.6). See Nunc Bulletins No. 7, 8, and 9.
What is the difference between certified and
non-certified MaxiSorp plates and modules?
Both of these surfaces are identical. The only difference between them is that
for the certified plates, a representative sample from each manufacturing lot
undergoes a Binding Capacity test. This test is an ELISA-like assay used in our
quality control laboratories to ensure binding capabilities. See Nunc Bulletin
No. 4.
What is the stability of the secondary amine CovaLink
surface with protein or DNA bound to the surface?
DNA, proteins or peptides bound to the CovaLink surface can be stored at 4°C
for up to one month.
What is the procedure for making the stock solution
of NHS-EDC as described in the CovaLink Users Manual?
The stock solution of NHS-EDC is made by mixing 0.2 M NHS with 0.2 M EDC at 1:1
ratio, making the final concentration of 0.1 M of each of NHS and EDC.
What are the features of the secondary amine
CovaLink surface?
CovaLink NH Modules are surface modified optically clear polystyrene modules in
strips of eight. They allow covalent binding of distinct groups of proteins,
peptides, oligosaccharides and DNA. This covalent-binding feature allows
orientation of the bound molecules such that the active site of the molecule is
available for biochemical activity. A key feature of the CovaLink is that the
polystyrene surface is grafted with secondary amino groups which serve as
bridges for covalent binding. The optically clear surface allows reading of
fluorescent or colorimetric signals.
What are some applications of CovaLink Modules?
What are some recommendations for improving binding
efficiency of proteins and DNA (oligonucleotides) using secondary amine CovaLink
modules?
Use a freshly made methylimidazole and carbodiimide condensing agents for
optimal covalent binding of both proteins and DNA (oligonucleotides). The
binding efficiency of single stranded oligo (a 5' phosphorylated end of a
single-stranded oligomer binds with a phosphoramidate bond) is about 8 - 10%
with a typical 25 base oligo.
Will Nunc-Immuno plates/modules fit into an
automated microtiter plate reader or washer?
Yes. All Nunc 96-well plates and frames for modules have a standard 96-well
footprint, 86 x 128 mm.
What are the advantages of one well geometry type
over another? Which is best for which application?
The following list describes the geometries of wells available for Nunc
Immuno-plates and modules.
Which components are included in the Nunc-Immuno
Wash Tubing Kit?
The tubing kit, Cat. No. 654569, consists of tubing, Y-shaped adaptor, and three
red clamps. Is the Eight Well Strip Cap compatible with
LockWell Modules?
No, due to the locking feature design of the LockWell Modules, the Eight Well
Strip Cap is not compatible and will not seal the LockWell Modules.
Is the Eight Well Strip Cap compatible with all
96-well MicroWell or Immuno Plates?
The Eight Well Strip Caps were designed to provide a positive seal for flat and
round bottom wells of 96 MicroWell or Nunc-Immuno plates. The Eight Well Strip
Caps are not compatible with C or V bottom wells of 96 MicroWell or Nunc-Immuno
Plates.
Is it possible to bind either single- or
double-stranded DNA to the MaxiSorp surface?
Single-stranded DNA can be adsorbed to MaxiSorp surface using approximately 10
µg ssDNA per ml PBS, pH 8.2. The stability is uncertain. Based on our
experience, ssDNA immobilized on the MaxiSorp surface is so loosely bound that
it is removed by stringent washing. Double-stranded DNA will not bind to the
MaxiSorp surface. DNA, however, can be covalently bound to NucleoLink Strips,
Cat. No. 248259.
What is the TSP (Transferable Solid Phase) and its
advantages?
The TSP is a disposable 96 pin device on which solid phase reactions can be
performed. It is available with a PolySorp and MaxiSorp surface. The pins are
coated by submerging in analyte solution contained in a 96-well plate. Washing
and reaction with succeeding antibody or streptavidin conjugates can be
performed by transferring the TSP into a washing tray. For hybridoma screening,
the TSP is available in a sterile version. Likewise, the TSP can be used for a
simultaneous detection of two different molecules in the same solution.
Advantages include: identical reaction times on all pins, no need for plate
washer/dispenser, and allows for a second solid phase reaction to be conducted
in a single 96-well plate.
What are some applications using the TSP
(Transferable Solid Phase)?
Reactions such as the ELISA can be performed on the TSP. The pins are coated by
submerging in the analyte solution contained in a 96-well plate. Washing and
reaction with succeeding antibody or streptavidin conjugates can be performed by
transferring the TSP into a washing tray or second 96-well plate filled with the
appropriate solution. The TSP is placed into a substrate solution until color is
observed and is then removed to ensure a simultaneous start and stop to the
enzymatic reaction. The TSP is available sterile for screening hybridoma cells
for the production and secretion of monoclonal antibodies. For radioimmune
assays, the TSP can be placed directly on X-ray film and exposed for several
hours. Only the tips of the pins should be incubated with the radiolabeled
reagent. The TSP can also be used with the OmniTray for performing dot blots and
for replicating bacterial clones from a 96-well plate. We offer a Tech Note,
Vol. 3 No. 24, that describes in situ screening of bacterial colonies using
Nunc-Immuno TSP.
What is the principle behind Elisa spot?
The Elisa spot technique was originally described by Sedgwick & Holt,
Journal of Immunological Methods, 57, 301, 1983. The basic principle is as
follows:
What length of peptide is ideal for binding to the
MaxiSorp surface and what are the detection limitations?
We have tested and found that a 3 amino acid peptide (Pro, Leu, Gly) cannot be
detected when passively adsorbed on the MaxiSorp surface. However, this peptide
can be detected when covalently immobilized using CovaLink NH Modules and
CovaLink NH2 Modules and Plates. Using covalent immobilization of small peptide
residues, one can obtain a better orientation of the molecule and reduced
problems with antibody recognition of the peptide due to masking of the epitope.
We have discovered that a 7 amino acid peptide from the MHC Class II antigen
can be detected when adsorbed on the MaxiSorp surface. We state that the
detection limitation using the MaxiSorp surface is between 3 and 7 amino acid
residues. One additional note is that detection is contingent upon the
orientation of the peptide when immobilized. If the active site is inactivated
or hidden at the site facing the solid phase, no detection signal is observed.